Mycobacterium ulcerans-Bordetella trematum chronic tropical cutaneous ulcer: A four-case series, Côte d’Ivoire

Background Chronic tropical cutaneous ulcers remain a neglected medical condition in West Africa, particularly Buruli ulcer, which is caused by mycolactone cytotoxin-secreting Mycobacterium ulcerans (M. ulcerans). Medical management of this highly debilitating and necrotising skin infection may be modified by colonisation and co-infection of the ulcer by opportunistic and pathogenic microorganisms, which considerably delays and increases the cost of treatment. Methodology/principal finding We diagnosed chronic tropical cutaneous ulcers in nine patients in Côte d’Ivoire using M. ulcerans-specific PCRs and culturomics. This revealed M. ulcerans in 7/9 ulcer swabs and 5/9 control swabs as well as an additional 122 bacterial species, 32 of which were specific to ulcers, 61 specifics to the controls, and 29 which were shared, adding 40 bacterial species to those previously reported. Whole genome sequencing of four Bordetella trematum (B. trematum) isolates in four Buruli ulcer swabs and no controls indicated cytolethal distending toxins, as confirmed by cytotoxic assay. Conclusions/significance In four cases of Buruli ulcer in Côte d’Ivoire, B. trematum was a co-pathogen which was resistant to rifampicin and clarithromycin, unmatching M. ulcerans antibiotic susceptibility profile and counteracting the current treatment of Buruli ulcer in West Africa and Australia. Thus, we report here chronic mixed M. ulcerans-B. trematum chronic tropical ulcer as a specific form of Buruli ulcer in West Africa.


Introduction
Tropical cutaneous chronic ulcers cause stigmatisation and are a significant handicap for the rural populations which mostly suffer from this this disabling condition.Nonetheless, it remains a neglected health problem in some West African countries [1].Several neglected tropical diseases (NTDs) are known to cause tropical cutaneous chronic ulcers including Buruli ulcer, yaws, leprosy, scabies and advanced lymphatic filariasis [2].Of these, Buruli ulcer is a necrotising and debilitating skin disease caused by Mycobacterium ulcerans (M.ulcerans) and reported by 34 countries, with West Africa and Australia the most affected [3].The disease progresses from cutaneous nodules to papules and oedema, eventually forming an ulcer with undermined edges [4].Further superinfection by pathogenic and opportunistic bacteria issued from the normal skin flora or the immediate environment, mainly Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P.aeruginosa), makes Buruli ulcer treatment challenging [5,6].These pathogens are further associated with delays in the healing process and significant increases in treatment costs [5][6][7].
In this study we investigate a series of collected swabs samples from patients in the M. ulcerans-endemic region of Yamoussoukro, Co ˆte d'Ivoire [8][9].Patients presented with tropical cutaneous chronic ulcer clinically diagnosed as Buruli ulcer.The results unexpectedly revealed a toxigenic Bordetella trematum (B.trematum) infection mixed with M. ulcerans in some patients, leading to the description of tropical cutaneous mixed bordetellomatosis-mycobacteriosis as a new clinical entity to be considered as a specific form of Buruli ulcer in West Africa.This also results in a complete, unprecedented characterisation of chronic cutaneous ulcer-associated B. trematum isolates.

Ethics statement
The study included patients (and parent in the case of infant) who gave oral consent for swabbing healthy skin.Accordingly, the study involved a non-invasive procedure and was approved by the National Committee for the Ethics of Life Sciences and Health of Co ˆte d'Ivoire under the reference 19-22/MSHP/CNESVS.

Patients
Nine patients consulted for chronic cutaneous ulcer as part of the routine medical practice at one of the authors' (SD) Buruli ulcer medical management centres in the Yamoussoukro

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Health District in Co ˆte d'Ivoire.These nine patients lived in the city of Yamoussoukro or surrounding rural areas in the Be ´lier district, an area that has been acknowledged for decades as being endemic for Buruli ulcer [9].Patient demographics and ulcer history are reported in Table 1.For each cutaneous ulcer, one swab contained in a transport medium (∑-Transwab, International Medical Products, Belgium) and one dry swab were gently applied by SD to the ulcer to perform routine and advanced microbiological investigations, seeking the microbial aetiology of the ulcer.With the oral consent of each patient, similar non-invasive swabbing was then performed on the skin surface contralateral to the ulcer, as an auto control clinical specimen.Collected swabs were anonymously coded, divided into two batches and placed in a well-sealed cooler containing dry ice.One batch was sent to the Institut Pasteur in Co ˆte d'Ivoire for first line diagnosis and the other batch was shipped at ambient temperature to the IHU-Me ´diterrane ´e Infection in Marseille, France for advanced diagnosis.

Molecular detection of M. ulcerans
Total DNA from all samples (lesion and auto control samples) was extracted using the QIAMP tissue kit on a QIAGEN-BioRobot EZ1 according to the manufacturer's instructions (Qiagen, Hilden, Germany).Extracted DNA was incorporated using real-time PCR (RT-PCR) to amplify the insertion sequences (IS2404 and IS2606) and the ketoreductase-B domain of the mycolactone polyketide synthase (KR-B) gene, using the RT-PCR reagents of the Roche PCR kit (Roche Diagnostics, Meylan, France) and the primers and probes previously described [10], in the CFX 96 real-time PCR detection and thermal cycler system (Bio-Rad, Marnes-la-Coquette, France) in the presence of negative controls.Samples were considered positive when the KR-B gene RT-PCR yielded a Ct < 40 cycles and at least one of the two insertion sequence PCRs yielded a Ct < 40 cycles, as previously described [11].

Swab culturomics
One hundred microliters of homogenized swab transport medium supernatant were serially diluted 10-fold up to 10 −10 in phosphate buffer saline (Thermo Fisher Scientific, Illkirch, France).Then, 50 μL of each dilution were plated onto Columbia agar enriched with 5% sheep blood (Becton Dickinson, Heidelberg, Germany) and incubated aerobically for 24 hours at 37˚C.Furthermore, 50 μL of each dilution plated onto Columbia agar enriched with 5% sheep blood plates were placed in a zip bag (Oxoid, Dardilly, France) supplemented with an anaerobic generator (Becton Dickinson) incubated in an anaerobic chamber (AES Chemunex, Combourg, France) at 37˚C for 48 hours.In parallel, 100 μL of such supernatant were also inoculated for 1 day, 3 days, 7 days and 10 days into BACT/ALERT flasks (bioMe ´rieux, Marcy PLOS NEGLECTED TROPICAL DISEASES l'Etoile, France) and YCFA liquid medium (DSMZ: Deutsche Sammlung von Mikroorganismen und Zellkulturen, Germany) (https://www.dsmz.de/microorganisms/medium/pdf/DSMZ_Medium1611.pdf),both supplemented with 2 mL of 0.2 μm-filtered rumen fluid and 2 mL of sterile defibrinated horse blood (bioMe ´rieux).After each incubation period, 100 μL of medium supernatant were collected and processed as described above for direct inoculation and aerobic and anaerobic incubation.All visible colonies were identified by Matrix Assisted Laser Desorption Ionization-Time of Flight mass spectrometry (MALDI-TOF-MS) (Microflex, Bruker Daltonik, Bremen, Germany) as previously described [12].Such identified B. trematum colonies were subcultured on 5% sheep blood Columbia agar at 37˚C for 24 hours for further analyses, as below.

B. trematum whole genome sequencing
Total genomic DNA was extracted from each of the four B. trematum isolates using the EZ1 protocol according to the manufacturer's instructions (Qiagen, Hilden, Germany).Briefly, 200 μL of B. tremarum suspension mixed with 200 μL of G2 buffer and 20 μL of proteinase K (Qiagen) was incubated at 56˚C for three hours.DNA extracted from 200 μL of mixture and eluted in a 50-μL volume was qualitatively and quantitatively evaluated using Qubit dsDNA

Genome annotation and comparative genomics
The quality and contiguity of assembled genomes was assessed using QUAST (5.0.2) [21] and the completeness of the assembled genome was assessed using Busco (v5.1.1)[22].Single-copy orthologous gene sets were searched, and gene content was compared with 569 genes in the Betaproteobacteria database (available at BUSCO v1 (ezlab.org)).Final genome assembly adjustments were performed by polishing using Pilon (v1.24) [23] with Illumina's own reads and the quality was then evaluated.Protein coding sequences (CDSs), tRNAs and rRNAs were predicted using Prokka [24] and the number of pseudogenes was calculated using the script get_pseudo.pl(VFDB: Virulence Factor Database (mgc.ac.cn)).Pathogenicity and antibiotic resistance analyses were conducted using the VFDB (Virulence Factors of Pathogenic Bacteria) [25] and CARD (Comprehensive Antibiotic Research Database) (The Comprehensive Antibiotic Resistance Database (mcmaster.ca))[26].

Patients
The study included nine patients with cutaneous chronic ulcers, seven of whom were males, with a mean age of 44.11 years (ranging from 14 to 83 years).All ulcers were primarily the lower extremities (7/9), and ulcers had been present for one month or more.Three patients had ulcers for more than one year and two were associated with the recurrence of lesions and diagnosed as Buruli ulcers more than ten years earlier.

M. ulcerans DNA detection
A total of 18 swabs (nine lesion swabs and nine lateral healthy skin control swabs) were tested for M. ulcerans in the presence of negative controls included in each PCR run remained negative for all targeted sequences, insertion sequences IS2404 and IS2606 and the ketoreductase-B (KR-B) gene targeted for identification of M. ulcerans.IS2404 was detected in all lesion swabs, KR-B was detected in seven and IS2606 in three of them.However, IS2404 and KR-B were detected in five control swabs and IS2606 in two control swabs.According to the previously defined criteria, 12 (66.66%)were positive for M. ulcerans including seven with positive lesions and five for lateral controls (Table 2).Four of seven patients with M. ulcerans-positive chronic ulcer also harboured M. ulcerans in the auto control swab.

Swab culturomics
Analysis of the microbiological composition of the swabs taken from the chronic ulcer samples of the nine patients revealed 99 bacterial isolates belonging to 61 different species, of which

Comparative microbiology
A total of 122 different bacterial species were isolated and identified from chronic ulcer and control swabs.Thirty-two species were specifically isolated from chronic ulcer swabs, 61 from control swabs and 29 species were common to both types of swabs (Fig 2 ).A comparison of the bacterial repertoire between the two types of swabs revealed that B. trematum and A. faecalis were significantly associated with chronic ulcer swabs (p value = .025,Fisher's exact test) and E. aurantiacum (p value = .04,Fisher's exact test) was significantly associated with control swabs.B. trematum and A. faecalis microorganisms were isolated from four of the seven documented M. ulcerans Buruli ulcer swabs and not from the healthy skin control swab.In particular, the B. trematum strain CI016 was isolated from a left ankle swab from a 16-year-old male, along with 13 other bacterial species (Fig 3A, Table 3).B. trematum strain CI 040 was isolated from swab from a reopened scar on the left elbow of a 40-year-old man, along with five other bacterial species (Fig 3B , Table 3).B.   3).

B. trematum antimicrobial susceptibility testing
Six B. trematum isolates, including four isolates recovered in this study and two isolates previously isolated at the Nice hospital laboratory (strains Archet 1 and 2), yielded a similar pattern of in vitro susceptibility and resistance to 35 antibiotics (Table 4).As for beta-lactamines,

Cytotoxicity assay
Testing the B. trematum cytotoxic effect on Vero E6 cells indicated cytotoxic activity of B. trematum isolates on Vero E6 cells.The activity was almost similar for all isolates but with the a slightly higher cytotoxic activity was observed (Fig 4).

Genomic analyses
The four investigated B. trematum WGSs produced between one and six contigs with > 96% completeness, establishing the reliability of the datasets for downstream analyses (Fig A and  Specifically, a flagellar system associated with mobility in all genomes examined accounted for 34% to 39% of annotated virulence factors; in addition to pili/fimbriae-associated proteins, filamentous hemagglutinin and LPS O-antigen, involved in Bordetella adhesion, accounting for 25% to 31% of genes.All B. trematum isolates expressed eight genes (pagP, bplA, bplB, bplC, bplD, bplE, bplL and kdsA) encoding lipopolysaccharide biosynthesis.We also noted the presence of a gene encoding the cytolethal distending toxin B subunit (CdtB).All isolates contained genes involved in biofilm formation (adeF, adeG and adeH), antiphagocytic activity (algW, wcbT), efflux (mexA, mtrD), and immune evasion, invasion, iron and magnesium absorption and stress adaptation.Strains CI016 and CI070 had protease-related genes.A search for drug resistance genes revealed potential genes encoding resistance to fluoroquinolone, tetracycline, disinfectants and antiseptics in all strains and strain CI016 had a gene Sul2 encoding sulphonamide resistance.
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Discussion
The first cases of Buruli ulcer were reported by Sir Albert Cook in his notes at Mengo Hospital in Kampala, Uganda in 1896 [27].However, this tropical infection was formally described by Australian colleagues in the early 1950s, uniquely combining epidemiology, clinical and pathology features, and isolation through the culture of the acknowledged aetiological agent, M. ulcerans [28].The then routine usage of convenient PCR (instead of fastidious culture) for the diagnosis of Buruli ulcer slightly complicated the situation after it was reported the detection of M. ulcerans DNA on apparently healthy skin, emphasizing that a positive PCR detection of this opportunistic pathogen was interpretable only on skin lesions, chiefly chronic ulcers [11].In this study, we adopted RT-PCR diagnostic criteria more stringent that the WHO-recommended IS2404 insertion sequence positivity, adding positivity of IS2606 and KR-B gene, to confirm cases of Buruli ulcer.Stringoury was chosen to increase Buruli ulcer diagnostic specificity, with the risk to miss true cases of Buruli ulcer: two samples (CI032 and CI053) were declared negative using our criteria despite Ct values of 36 and 37 for the IS2404 sequence.These results could either indicate a low bacterial load or suggest that our criteria excluded borderline cases.Examination of the clinical context adds a further layer of complexity: patient CI032 presented a very old lesion, a reopening of the scar of an old confirmed Buruli ulcer, which could justify a low bacterial load; patient CI053 was in the early stages of the disease, which would also explain the low bacterial load.Further microbiological investigations identified a total of 25 different bacteria in cases of Buruli ulcer confirmed by PCR including S. aureus and P. aeruginosa, which have been convincingly associated with delayed healing of Buruli ulcer [5,7,29] (Table 5).
Using culturomics and auto control as previously reported in investigation of diabetic ulcers (as an example) [30], our study significantly expanded the bacterial repertory of Buruli ulcers confirmed by PCR, adding 40 bacterial species to the previous reports, revealing a previously unreported prevalence of B. trematum co-infection with M. ulcerans in this situation of tropical chronic cutaneous ulcers.B. trematum has been previously delineated as a unique bacterial species after a polyphasic investigation of ten isolates, including six isolates recovered from open wounds in patients, most of whom were European in origin [31].B. trematum was further isolated in thirteen patients presenting with chronic cutaneous ulcers sometimes extending to underlying tissues and bones, and most patients originated from European countries [32][33][34][35][36][37], the United States [38], Brazil [39], Argentina [40] and Japan [41].However, B. trematum has never been previously reported as a co-pathogen with M. ulcerans.

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Genomic analysis revealed virulence factors associated with colonisation (adhesin), biofilm formation and endotoxin production; all factors which may contribute to delayed Buruli ulcer healing, as previously reported for S. aureus or P. aeruginosa [5,6].Moreover, the four B. trematum isolates here recovered from M. ulcerans-positive chronic ulcers were all producing cytotoxin.Indeed, B. trematum is the only Bordetella species which encodes for cytolethal PLOS NEGLECTED TROPICAL DISEASES distending toxin, a toxin that causes DNA damage and cell cycle arrest also encoded by other gram-negative pathogens such as Haemophilus ducreyi [42][43].Lastly, our observations may be relevant for the medical management of Buruli ulcer patients in West Africa as the natural in vitro antibiotic susceptibility profile of B. trematum sharply differed from the one of M. ulcerans [44].Here, B. trematum isolates were consistently resistant to rifampicin and clarithromycin, the WHO-endorsed antibiotic combination for treating Buruli ulcer in West Africa; while previously reported resistance to some fluoroquinolones [32,33,35] would have compromised success when treating Buruli ulcer (mixed with B. trematum) in Australia.Further, B. trematum isolates were resistant to aminoglycosides, as previously reported [35,45], whereas aminoglycosides were used until recently in West Africa to treat Buruli ulcer [46,47].
Present study offers one more example of polymicrobial chronic tropical ulcer: our study echoes previous microscopic observations of mixed spirochetes and fusobacteria in tropical ulcers in four different tropical countries (Zambia, Gambia, southern India and Papua New Guinea); recently complemented by metagenomic confirmation of Fusobacterium necrophorum in samples from Papua New Guinea [48,49].Understanding microbial diversity in tropical ulcers may offer valuable insights into the diagnosis and management of Buruli ulcer, particularly in cases where co-infections may play a role.
In conclusion, we propose that a mixed M. ulcerans-B.trematum chronic tropical cutaneous infection is one form of Buruli ulcer warranting specific attention and diagnosis.In this perspective, further research incorporating a larger number of chronic tropical ulcers cases in different Buruli ulcer endemic West African countries is needed to understand the actual contribution of toxin-secreting B. trematum strains to this situation and the opportunistic role of toxin-secreting B. trematum strains as co-pathogens with M. ulcerans, taken into consideration the unique antibiotic susceptibility profile of this bacterium; in particular in patients who fail to respond to initial M. ulcerans treatment.
trematum strain CI070 was co-isolated with 24 bacterial species from a swab of a right foot in a 70-year-old man (Fig 3C, Table 3).B. trematum strain CI083 was isolated from a swab of the right foot of an 83-year-old man, along with eight other bacterial species (Fig 3D, Table

Fig 4 .Fig 5 .
Fig 4. trematum cytotoxicity for Vero E6 cells.B. (a): Confluent Vero E6 cells were incubated with a bacterial suspension (MOI = 100).After two hours of incubation, the CellTiter-Glo kit was used for cytotoxicity testing.For the negative control of cell death, cells were incubated only with 1x minimum Eagle's medium (MEM) with 4% heat-decomplemented foetal calf serum and 1% glutamine.Then a Sarcina ventriculi known to be devoid of cytotoxic activity was used as a control of negative cytotoxic activity.Digitonin was used as a positive control for cytotoxic activity.(b): Confluent Vero E6 cells were incubated with filtered and lyophilised bacterial supernatant resuspended in 1x minimum Eagle's medium (MEM) with 4% heat-decomplemented foetal calf serum and 1% glutamine.After two hours of incubation, the CellTiter-Glo kit was used for cytotoxicity testing.For the negative control of cell death, cells were incubated only with 1x minimum Eagle's medium (MEM) with 4% heat-decomplemented foetal calf serum and 1% glutamine.The TCSB medium used for bacterial culture, treated under the same conditions, was used to control the cytotoxic activity of the medium.This activity was then deduced from the activity of B. trematum isolates.Digitonime was used as a positive control for cytotoxic activity.https://doi.org/10.1371/journal.pntd.0011413.g004

Table 1 .
Patient demographics.Timescale refers to delay for ulcer evolution.NA, not available.

Table 2 . Buruli ulcer PCR test results.
Sample are considered positive for M. ulcerans if the KR-B gene was detected with a Ct < 40 cycles and at least one of the two insertion sequence PCRs resulted in a Ct < 40 cycles and KR-B was detected with a Ct < 40 cycles.

Table 3 . Bacterial species isolated from ulcer swabs (and not from healthy skin swab) positive for M. ulcerans (PCR detection) and for B. trematum (culture detec- tion), in Co ˆte d'Ivoire.
Patients coded as CI0X.